The movement of a molecule with a net charge in an electric field is known as electrophoresis. Electrophoresis is a powerful means of separating biological molecules such as proteins, peptides, oligonucleotides, RNA and DNA. The velocity of migration (ν) of a molecule in an electric field depends on the electric field strength (E), the net charge of the protein (z), and the frictional coefficient (f), as shown in the equation:   v  =            E      ⁢                           ⁢      z        f  
Two primary separating mechanisms exist: (i) separations based on differences in the effective charge of the analytes, and (ii) separations based on molecular size. Separations based on differences in the effective charge are usually limited to low or moderate molecular weight molecules since high molecular weight molecules have effective charges that are similar, making it difficult to separate them. Separations based on molecular size are generally referred to as molecular sieving and are conducted using gel matrices that have controlled pore sizes. In these separating systems, if the effective charges of the analytes are the same, the separation results from differences in the abilities of the different sized molecular species to penetrate through the gel matrix. Smaller molecules move more quickly relative to larger ones through a gel of a given pore size.
In theory, separation of different proteins could be achieved readily in free solution provided that the molecules differed sufficiently in their charge densities. In practice, separations in free solutions are difficult to achieve because the heat produced during electrophoresis can cause convection disturbances which distort the protein bands. As a result, band broadening occurs and protein resolution is compromised. For this reason, electrophoresis in free solution is rarely practiced.
Various support media are used to minimize convection and diffusion, such as paper or cellulose acetate, agarose, starch and polyacrylamide. Paper, cellulose acetate, and similar porous materials are relatively inert, serve mainly for support and to minimize convection, and separation of proteins is based largely on the charge density of the proteins at a specific pH.
Starch, agarose and polyacrylamide gels are the preferred media for conducting electrophoretic separations. These gels not only minimize convection and diffusion, but also actively participate in the separation process. More specifically, these materials provide a restrictive medium where molecular sieving occurs, and provides separation on the basis of both charge density and molecular size. Polyacrylamide-based gels are widely used because they are chemically inert, have excellent sieving characteristics, and have high resolution capabilities. However, polyacrylamide-based gels have high viscosity, may form air bubbles that affect separation, have a short shelf life, are neurotoxic, and are cumbersome to prepare.
Electrophoresis may be carried out using slab gels, which typically run several samples in parallel lanes. Large samples of DNA are required, and detection of the DNA typically occurs after the electrophoretic separation is complete. Advantages of this technique include the ease of post-separation sample collection, the ability to compare results of multiple samples on the same gel, and the ability to separate large amounts of DNA. However, slab gel electrophoresis can be labor-intensive and slow.
Alternatively, capillary electrophoresis (CE) permits rapid, high resolution separation of molecules. CE typically involves the separation of charged molecules in a narrow capillary with on-line detection by either absorbance or laser-induced fluorescence. Typically, CE employs fused silica capillary tubes whose inner diameters are between 50 microns to 200 microns, and have lengths ranging from 10 cm to 100 cm or more. These capillaries can be coated , typically with polyacrylamide, or left uncoated. The coating prevents electroendoosmotic flow, a phenomenon which frequently occurs in electrophoretic separations of solute ions dissolved in a solvent or solvent system. This phenomenon causes bulk flow of the solvent system in response to the applied electric field. The bulk flow impairs the separation of solutes since it causes mobilization of all solutes at a common rate as part of the solution in which they are dissolved. This effectively shortens the path of travel attributable to electrophoresis, and thereby lessens the degree of electrophoretic separation for a column of a given length.
Crosslinked polymers, such as polyacrylamide and other high viscosity media, are not suitable for CE since the high viscosity makes replacement of the gel in the capillary an impractical matter. Separation also takes a longer period of time using crosslinked polymers when compared to low viscosity media.
Linear polyacrylamide (“LPA”) is a highly effective separation medium that has excellent sieving properties due to its entanglement structure. Furthermore, the absence of crosslinking in a linear polymer gives it lower viscosity. In order to use LPA in CE, the capillary inner wall must be coated with a hydrophilic polymer to suppress electroendoosmotic flow in order to achieve expected separation performance. Coating the inner surface of capillary walls to prevent electroendoosmosis is known. For example, U.S. Pat. No. 5,545,302 discloses the suppression of electroendoosmotic flow in CE by the use of amine-derivatized polymers. U.S. Pat. No. 5,552,028 discloses the suppression of electroendoosmostic flow in CE by the use of N,N′-disubstituted or N-monosubstituted polyacrylamides, which have self-coating properties. These amine-derivatized polymers were prepared by modifying the functional groups of a pre-existing homopolymer to impart the desired self-coating characteristic. U.S. Pat. No. 4,997,537 discloses a silane-derivatized coating covalently bonded to the inner surface of a microcapillary wall, a thin layer of a hydrophilic polymer absorbed on the layer of the coating material, and a polyacrylamide gel filling the tube.
Other polymers, such as poly(ethylene oxide), poly(dimethylacrylamide) (U.S. Pat. Nos. 5,567,292 and 5,552,028), and poly(vinylpyrrolidone) (Gao et al., Analytical Chem., 1998, 70, 1383-1388) are able to suppress electroendoosmotic flow during DNA separation in uncoated capillaries. However, none of these polymers has been shown to surpass the performance of linear polyacrylamide in the aspects of DNA sequencing read length and capability of high speed separation at elevated temperature.
The use acrylamide derivatives to synthesize polymeric gel matrices is known (Sassi et al., Electrophoresis, 1996, 17, 1460; U.S. Pat. No. 5,470,916; and Lindberg et al. Electrophoresis, 1997, 18, 2909). Gel Matrices synthesized from these acrylamide derivatives usually have significant physical, chemical, and electrophoretic properties from polyacrylamide.
The use of copolymers in electrophoresis media to enhance performance are known. For example, U.S. Pat. No. 4,997,537 discloses the use of a copolymer of polyacrylamide and a crosslinker, such as N,N′-methylenebisacrylamide, in microcapillary gel electrophoresis. U.S. Pat. Nos. 4,948,480 and 5,149,416 disclose gel electrophoresis media composed of a water-soluble copolymer of an acrylamide monomer with another comonomer that facilitates crosslinking via a crosslinking agent. U.S. Pat. No. 5,759,369 discloses an electrophoresis separation medium composed of a copolymer containing a linear hydrophilic polymer segment having uniform segment length and hydrophobic polymer segments at each end. These copolymers may have one of the following structures: i) comb or tuft copolymer structure; ii) block copolymer structure; and iii) star copolymer structure, but none are random copolymers. Liang et al. (Electrophoresis, 1998, 19, 2447-2453) discuss capillary electrophoresis using a non-crosslinked triblock polymer.
Despite the great amount of effort which has gone into improving conventional electrophoresis separation media, there is a need for new polymeric media that incorporate specific properties to overcome general problems associated with electrophoretic separations, and to enhance electrophoresis separation performance in various applications. What is also desirable is an electrophoresis gel medium for coated and uncoated capillaries that is capable of resolving DNA fragments comprising 500 to 1000 base pairs.